Age-dependent Powassan virus lethality is linked to glial cell activation and divergent neuroinflammatory cytokine responses in a murine model

ABSTRACT Powassan virus (POWV) is an emergent tick-borne flavivirus that causes fatal encephalitis in the elderly and long-term neurologic sequelae in survivors. How age contributes to severe POWV encephalitis remains an enigma, and no animal models have assessed age-dependent POWV neuropathology. Inoculating C57BL/6 mice with a POWV strain (LI9) currently circulating in Ixodes ticks resulted in age-dependent POWV lethality 10–20 dpi. POWV infection of 50-week-old mice was 82% fatal with lethality sequentially reduced by age to 7.1% in 10-week-old mice. POWV LI9 was neuroinvasive in mice of all ages, causing acute spongiform CNS pathology and reactive gliosis 5–15 dpi that persisted in survivors 30 dpi. High CNS viral loads were found in all mice 10 dpi. However, by 15 dpi, viral loads decreased by 2–4 logs in 10- to 40-week-old mice, while remaining at high levels in 50-week-old mice. Age-dependent differences in CNS viral loads 15 dpi occurred concomitantly with striking changes in CNS cytokine responses. In the CNS of 50-week-old mice, POWV induced Th1-type cytokines (IFNγ, IL-2, IL-12, IL-4, TNFα, IL-6), suggesting a neurodegenerative pro-inflammatory M1 microglial program. By contrast, in 10-week-old mice, POWV-induced Th2-type cytokines (IL-10, TGFβ, IL-4) were consistent with a neuroprotective M2 microglial phenotype. These findings correlate age-dependent CNS cytokine responses and viral loads with POWV lethality and suggest potential neuroinflammatory therapeutic targets. Our results establish the age-dependent lethality of POWV in a murine model that mirrors human POWV severity and long-term CNS pathology in the elderly. IMPORTANCE Powassan virus is an emerging tick-borne flavivirus causing lethal encephalitis in aged individuals. We reveal an age-dependent POWV murine model that mirrors human POWV encephalitis and long-term CNS damage in the elderly. We found that POWV is neuroinvasive and directs reactive gliosis in all age mice, but at acute stages selectively induces pro-inflammatory Th1 cytokine responses in 50-week-old mice and neuroprotective Th2 cytokine responses in 10-week-old mice. Our findings associate CNS viral loads and divergent cytokine responses with age-dependent POWV lethality and survival outcomes. Responses of young mice suggest potential therapeutic targets and approaches for preventing severe POWV encephalitis that may be broadly applicable to other neurodegenerative diseases. Our age-dependent murine POWV model permits analysis of vaccines that prevent POWV lethality, and therapeutics that resolve severe POWV encephalitis.

F laviviruses (FVs) are a family of enveloped, positive-strand RNA viruses that cause human disease, and numerous FVs are transmitted by arthropod vectors (1).Tick-borne FVs cause ~15,000 annual cases of severe encephalitis and include the tick-borne encephalitis virus (TBEV) in Eurasia and the Powassan virus (POWV) in North America (2).POWV (strain LB) was first isolated in 1958 from a human encephalitis case in Powassan Ontario, Canada (3).POWVs are emerging in the Northeastern United States due to the expansion of animal reservoirs, tick vectors, and increasing clinical awareness of human POWV infections (2,(4)(5)(6)(7)(8)(9)(10)(11)(12)(13).In endemic US states, the seroprevalence of POWV is between 0.7% and 6.1%, with fewer clinical cases suggesting that infections are largely asymptomatic (2,14).POWV is present in tick saliva, transmitted in as little as 15 minutes of a tick bite (2,11,15), and severe cases cause 10% fatal encephalitis and long-term neurologic sequelae in 50% of survivors (2,16).POWV infects all age groups, with limited data suggesting that lethality and severe neurologic sequelae are increased in patients >60 years of age, similar to clinical outcomes of TBEV infection (2,13,17,18).POWV infections are biphasic with an initial acute febrile illness 1-3 weeks after a tick bite followed by weeks of wide-ranging CNS manifestations (2,16,17).Fatal human POWV encephalitis presents with severe CNS damage, brainstem involvement, inflammation, and gliosis in the cerebral cortex, without histologic evidence of systemic infection (2,16,17).Currently, there are no clinically approved POWV therapeutics or vaccines.
In comparison to TBEV, the CDC reported that 8% of POWV patients were <18 years old, and ~50% of patients were >60 years old (2).In a cohort of 99 POWV cases, the median age was 62 years and all fatalities (11%) occurred in patients older than 50 years of age (7).Among 14 cases of POWV encephalitis in New York, 72% of patients were >60 years old, with five fatalities in patients > 60 years of age, and neurologic deficits in all survivors (14,17).Autopsies revealed reactive gliosis, increased microglia, and necrotizing CNS inflammation consistent with acute meningoencephalitis (2,14).These findings suggest age as a factor in the severity of encephalitis caused by TBEV and POWV.
Increases in inflammatory Iba1 + staining, reflective of reactive microglial nodules and microgliosis (75,76), were apparent in all CNS regions of 10-and 50-week-old mice (Fig. 3A).Iba1 + staining of the pons increased from 5 to 15 dpi, as assessed by ImageJ quantification (10 areas per mouse, n = 4; Fig. 3B).Increases in reactive glia from 5 to 15 dpi were also observed and quantified in the pons of LI9-infected 20-to 40-week-old mice (Fig. S1 and S3).Similar analysis and quantification revealed increases in GFAP + astrocytes from 5 to 15 dpi in 10-and 50-week-old POWV-infected mice (Fig. 4).These results demonstrate that POWV is neuroinvasive, causing rapid CNS neuronal damage by 5 dpi, with dramatic increases in reactive glial cells from 5 to 15 dpi in the CNS of all age mice.
Mice of all ages that survived lethal infection (30 dpi) had comparable viral loads that were near the level of detection (Fig. 7A).In situ hybridization (ISH) of positive-stranded POWV RNA in the CNS (27,85,86) failed to detect POWV RNA in the CNS 5 dpi, and detected sporadic POWV RNA in single-cell foci dispersed throughout the brains of 10and 50-week-old mice, 10-15 dpi (Fig. 7; Fig. S4).ISH staining of POWV RNA was notably increased in the cerebral cortex (Fig. 7B) and midbrain (Fig. S4) of

DISCUSSION
Severe encephalitis and long-term neurologic damage are associated with human POWV infections (2,16), but mechanisms of POWV neuropathology and lethality remain largely unknown.Despite a lack of systemic infection or peripheral disease, POWV patients display profound cerebellar and brainstem involvement, characterized by neuronal loss, microgliosis, inflammatory infiltrates, and POWV RNA in the CNS (2,16).Data availa ble from a limited number of human POWV cases indicates that many lethal POWV infections occur in individuals >60 years of age (2, 7, 14, 17) and are consistent with the lethality of TBEV infections (38,39) and the age-dependent severity of WNV encephalitis (79,(93)(94)(95).Models of POWV infection were previously studied in peripherally inoculated 5-to 14-week-old mice primarily using murine brain neuroadapted LB and SP strains (2,12,24,25,27,29,30).In these studies, lethality varied by strain, dose, kinetics, and suggested requirements for tick saliva in directing neurovirulence (29,36).Here, we assessed the age-dependent lethality of POWV in 10-to 50-week-old mice using a LI9 POWV strain present in circulating Ixodes ticks that was directly isolated and passaged in VeroE6 cells (21,42).Footpad inoculation of POWV LI9 into C57BL/6 mice resulted in 82% lethality in 50-week-old mice, with lethality sequentially reduced with age to 7.1% in 10-week-old mice (Fig. 1C and F).Murine lethality was not sex linked, and a minimal infectious dose of LI9 was 80% fatal in 50-week-old mice (Fig. 1E through G).These findings establish the age-dependent lethality of POWV LI9 in a murine model that reflects the severity of human POWV disease in the elderly (13).
Similar to CNS degeneration observed at autopsy of POWV patients, histopathology of LI9-infected mice revealed spongiform CNS lesions in cerebellar and hindbrain regions of the pons, medulla, and brainstem that were accompanied by neuronal depletion and the presence of reactive microglial nodules (2,13,16,65).We analyzed four time points (5, 10, 15, and 30 dpi) post-POWV infection for CNS histopathology, viral load, glial, and cytokine responses across murine age.At 5 dpi, there were no clinical signs of POWV infection, and CNS viral loads were at minimal levels or undetected by qRT-PCR or ISH.Yet, the CNS of all age mice contained focal spongiform lesions, neuronal depletion, and reactive CNS resident glial cells 5 dpi.This suggests that early damage to the CNS following POWV infection is age-independent, but fails to reveal potential causes of POWV lesions (neuroinvasion, apoptosis, glial cell phagocytosis, etc.) or if neurodegener ative or cognitive deficits are common to all POWV survivors (2,7,9,14,16,17,96).
We found high POWV loads in the CNS of all age mice at 10 dpi, before most mice show clinical neurologic signs.Comparably high CNS viral loads 10 dpi indicate that POWV neuroinvasion and replication in the CNS are age-independent and distinct from subsequent late-stage age-dependent lethality.CNS histopathology 10 dpi revealed a marked reduction in spongiform lesions in mice of all ages, and the increased presence of reactive microglia/macrophages and astrocytes (44-49, 58, 59, 62, 75, 76, 91, 97, 98).These findings are consistent with CNS cell proliferation, tissue replacement, and repair that occurs 2-10 days after CNS damage and is directed by reactive glial and neuro nal progenitor cells (68)(69)(70).Thus, at 10 dpi, glial cell proliferation, inflammation, and tissue repair in the CNS occur simultaneously with high levels of POWV replication, and the consequence of these opposing responses appears pivotal in determining murine survival or lethality.
Despite age-dependent differences in POWV lethality, in all age mice clinical signs and lethality were observed between 10 and 20 dpi (1/49 fatalities occurred 9 dpi).Analysis of mice at an acute lethal stage of infection, 15 dpi, revealed focal spongiform vacuoles in all aged mice with increased levels of reactive glia cells throughout the CNS.At 15 dpi, a reduction in CNS viral loads was noted in 10-to 40-week-old mice, while POWV loads in the CNS of 50-week-old mice remained undiminished and identical to In all mice surviving POWV infection (30 dpi), we found POWV RNA levels near background while spongiform damage and reactive glial cells were persistent and pervasive in the CNS.Reactive glial cells were increased in all age mice 5-30 dpi suggesting that CNS repair and tissue remodeling are ongoing during early and acute stages of POWV infection as well as in survivors 30 dpi (68)(69)(70).Our findings suggest that persistent CNS POWV loads and reactive glial cell responses may contribute to age-dependent POWV lethality as well as long-term neurodegenerative damage or CNS inflammation in mice.This is consistent with 10% lethality and severe long-term neurologic sequelae observed in ~50% of surviving POWV patients (2,7,14,17).It remains to be determined whether POWV-directed CNS pathology in mice causes long-term cognitive sequelae and whether cognitive changes are age independent.
In murine WNV studies, kinetic CNS analysis and glial cell responses remain to be factored into age-dependent severity, with suggested increases in CD4/CD8 T cells and CD8 reactivation reported to distinguish WNV clearance in young versus aged mice (79, 93, 95, 99, 104-106).By contrast, we observed few CD4/CD8 T-cell infiltrates in the CNS, and no discernible differences in T-cell infiltrates in POWV-infected 10-versus 50-weekold mice (79,95).Our findings associate glial cell activation with kinetic changes in CNS cytokine responses directing a neuroinflammatory state that results in age-dependent POWV lethality and long-term CNS pathology in survivors.It remains to be determined whether cytokine responses of young mice reveal potential therapeutic targets and approaches for preventing severe/lethal disease in aged mice, and POWV infections of the elderly.Neuronal damage directs CNS tissue regeneration through neuronal stem cell (NSC) and microglial proliferation; however, reactive glial cells can also inhibit CNS regener ation (101,(109)(110)(111). Aging is known to decrease regenerative NSCs and increase senescent microglia and T cells (108,112,113) that persistently secrete pro-inflammatory cytokines (101,110,112,(114)(115)(116) causing an age-dependent senescence-associ ated secretory phenotype (SASP) (2,13,16,(117)(118)(119).In the CNS, the expression of IFNα, IL-2, and IL-12 is linked to experimental autoimmune encephalitis (EAE), multiple sclerosis (MS), Alzheimer's disease (AD), and Parkinson's disease (PD) (59,84,87,117,118,(120)(121)(122) through amplified Th1 responses (51,52,55,56,87,123).In AD and PD, age-dependent cognitive deficits are also associated with the accumulation of senescent NSCs and microglia in a severe proinflammatory state (112,114).CNS cytokine responses of POWV-infected 50-week-old mice reflect induced senescent cell cytokines (TNFα, IL6, and IL-1β) that may contribute to SASP.Anti-inflammatory steroids inhibit IL-12 and Th1 responses, and either blocking IL-12 or enhancing Th2 responses reduces AD pathology (87).There is a single report that five corticosteroid-treated POWV patients survived, while POWV was lethal in 5/5 untreated patients > 60 years of age (14).This anecdotal finding suggests the potential for steroids to suppress late neuroinflammatory POWV damage, and our lethal POWV murine model provides a means for assessing the efficacy of anti-inflammatory approaches.However, whether CNS repair, senescence or inflammatory glial cell responses contribute to age-dependent POWV lethality remains to be examined.
Our findings reveal a novel age-dependent murine model of lethal POWV encepha litis and long-term neurologic damage in survivors.Histopathology of POWV-infected aged murine brains mimics POWV-induced CNS pathology in fatal human cases (16) and long-term spongiform damage observed in survivors irrespective of age (2,16).Our results associate Th2-type cytokine responses with POWV clearance and survival in young mice, and Th1-type cytokine responses and increased CNS viral loads with the lethality observed in 50-week-old mice.Our studies suggest roles for persistently activated glial cells, neurodegenerative M1/Th1 type cytokine responses, and failed POWV clearance with lethal outcomes in aged mice.This murine model provides a basis for defining the protective responses of young mice and age-dependent causes of POWV lethality.Further studies of young versus aged mice are likely to suggest therapeutic targets and approaches for resolving age-dependent POWV lethality in elderly patients and may be applicable to other age-dependent viral and chronic neurodegenerative diseases.

Biosafety and biosecurity
Animal research was performed in accordance with institutional guidelines using approved experimental protocols, and supervised by the Institutional Biosafety and Institutional Animal Care and Use Committees at SBU. Animals were managed by the SBU Division of Laboratory Animal Resources, which is accredited by the American Association for Accreditation of Laboratory Animal Care and DHHS, and maintained in accordance with the Animal Welfare Act and DHHS "Guide for the Care and Use of Laboratory Animals." Veterinary care was directed by full-time resident veterinarians accredited by the American College of Laboratory Animal Medicine.POWV murine infection experiments were performed in an animal biosafety level 3 facility (The Laboratory of Comparative Medicine, Stony Brook University).

Murine inoculation
C57BL/6J mice (10-50 weeks old) were purchased from Jackson Laboratory.Mice were anesthetized via intraperitoneal injection with 100 mg of ketamine and 20 mg of xylazine per kilogram of body weight.Animals were infected by subcutaneous footpad injection with up to 2 × 10 3 FFU POWV or PBS in a volume of 20 µL.Mice were weighed daily and evaluated for signs of clinical neurologic disease.Mice that reached humane endpoints including non-responsiveness or severe neurologic signs (hindlimb flaccid paralysis, ataxia, inability to self-right) were euthanized from 1 to 20 dpi or at predetermined 5, 10, 15, and 30 dpi time points (64).Murine POWV inoculation and lethality analysis were replicated in at least three independent experiments.
Histopathology scoring of H&E stained age-matched brain tissues was performed blinded to age and experimental group.Brain regions were scored on a scale of 0-4 for spongiform encephalopathy, microgliosis, and neuronal necrosis.Scores define localized severity of pathology: (0) baseline of age-matched control brain staining in select region, (1) localized lesion, (2) multiple localized lesions, (3) lesions spread throughout most of selected region, and (4) lesions uniformly spread throughout select region (71).ImageJ quantification of Iba1 immunostaining of 50-week-old mouse brains versus age-matched mock-infected controls: relative percent area of Iba1 + pixel intensity for 10 regions was determined per POWV-infected brain and compared to mock-infected controls.Brain section quantitation is derived from ImageJ analysis of at least 10 brain locations from each of four individual mice.
For RNA in situ hybridization (ISH), FFPE brain tissues were sectioned (5 µm thick ness), xylene deparaffinized, and antigen retrieval treated in a decloaking chamber for 1 h.POWV RNA was detected using the RNAScope Universal AP assay (Advanced Cell Diagnostics, Inc.) according to the manufacturer's protocol.RNAScope 2.3 VS probe V-Powassan (catalog #415641) was used to detect genomic POWV RNA (27).ISH-probed slides were analyzed using QuPath software.

RNA extraction and qRT-PCR analyses
Age-matched mock or POWV-infected mice were euthanized and brains were harves ted in TRIzol LS Reagent (Invitrogen) homogenized and RNA purified according to the manufacturer's protocols.RNA was processed using Monarch RNA Cleanup Kit (NEB T2030L) and quantified on a Nanodrop Spectrophotometer 2000.To define viral loads and inflammatory transcripts in POWV-infected brains, qRT-PCR was performed  (21,42,115).cDNA synthesis was performed using random hexamer priming of a Transcriptor first-strand cDNA synthesis kit (Roche) as previously described (21,42,60).Viral RNA levels were assayed using NS5-specific LI9 primers (Table 2) and compared to a standard curve of serially diluted POWV RNA.qRT-PCR transcript primers were designed using the NCBI gene database, with 60°C annealing profiles (Table 2).Transcript levels were analyzed in triplicate from n = 3 age-matched control or n = 3 POWV-infected mice using PerfeCTa SYBR green SuperMix (Quanta Biosciences) on a Bio-Rad C1000 Touch system with a CFX96 optical module (Bio-Rad).Responses were normalized to internal GAPDH mRNA levels, and the fold induction was calculated using the threshold cycle (2 -ΔΔCT ) method for differences between age-matched mock and POWV-infected RNA levels at each time point post-infection.

Statistical analysis
The statistical significance of the results was determined using Prism 6 software (GraphPad Software, Inc.; https://www.graphpad.com).Statistical analysis for individual experiments is presented in the figure legends.The infection of young vs aged mice has been replicated in at least three independent experiments and additionally by determining the minimal POWV dose required for lethality.Histopathology analyses are compared across groups using one or two-way ANOVA with replicate sample sizes having n = 4 mice/age and time points in all figures (71).The number of animals for the histopathology study has been calculated based on the minimal number needed to achieve statistical significance.Based on prior studies, the values obtained for the mean and standard deviation for the control and experimental groups suggest that four animals per group would provide 80% power (Alpha of 0.05) to detect a change of 2 in histological scores.Kaplan-Meier curves were analyzed by a log rank test.P values of less than 0.05 were considered statistically significant.

FIG 4
FIG 4 POWV infection causes astrocyte activation in mice.(A and B) C57BL/6 10-and 50-week-old mice were footpad inoculated with 2 × 10 3 FFU of POWV LI9 or mock infected with PBS.Brains were harvested 5, 10, and 15 dpi, sectioned, and anti-GFAP immunostained to identify activated astrocytes.(A) Representative images of astrocytosis in the pons, medulla, brainstem, cerebellum, midbrain, and cerebral cortex of POWV-versus mock-infected 50-week-old brains are presented.(B) ImageJ quantification of GFAP immunostaining: relative percent area of GFAP + pixel intensity was determined for 10 CNS regions of 10-and 50-week-old mice (n = 4), 5, 10, and 15 dpi, vs mock-infected controls.Data are presented as means with SEM and individual data point comparisons to mock-infected controls were performed by two-way ANOVA analysis.Asterisks indicate statistical significance (*P < 0.05).

FIG 7 (Full 12 FIG 8
FIG 7 (Continued) group reflect an n = 4 following POWV infection and n = 11 for mock-infected controls.Data are presented as means and each dot represents an individual mouse.Individual data point comparisons were performed by two-way ANOVA analysis of log 10 transformed values.Asterisks indicate statistical significance (**P < 0.001, ***P < 0.0001).(B) Brains from POWV-infected or mock-infected 10-and 50-week-old mice were harvested at 5, 10, and 15 dpi, and genomic POWV RNA in the CNS was detected by in situ hybridization (ISH) with POWV RNAscope probe-red (ACD).Representative images of POWV RNA ISH in the cerebral cortex of POWV-infected (n = 3) and mock-infected 50-week-old mice (n = 1) are presented.Black arrows indicate representative cells positive for genomic POWV RNA by ISH.

TABLE 1
Neutralizing antibody titers of 10-to 50-week old mice following POWV LI9 infection 50-week-old brain sections 15 dpi.Collectively, these findings demonstrate that POWV LI9 neuroinvasion is age independent, with high viral loads in the CNS of all age mice 10 dpi.However, the dramatic reduction in POWV viral load in younger mice from 10 to 15 dpi was not observed in 50-week-old mice, where high CNS viral loads occur coincident with acute infection and 82% lethality.

FIG 8
(Continued) levels.At each time point, age group-infected or mock-infected age-matched control is presented (n = 3).Data are presented as means with SEM and each dot represents an individual mouse.Individual data point comparisons were performed by two-way ANOVA analysis.Asterisks indicate statistical significance (*P < 0.05; **P < 0.01).(B)A summary of the fold induction of 10-and 50-week-old CNS cytokine and chemokine responses over age-matched controls 15 dpi is presented.Findings are sorted by the fold induction of 50-week/10-week-old CNS responses with fold differences and a heat map that segregates age-dependent responses.CNS viral loads found in moribund 50-week-old mice.These findings suggest potential age-dependent differences in POWV clearance from the CNS, replication rates, CNS damage (POWV or immune mediated), or discrete CNS repair responses, as mechanisms contributing to persistently high CNS viral loads 15 dpi, and age-dependent lethality.Additional kinetic analysis of CNS viral loads between 10 and 15 dpi may clarify the role of sustained POWV CNS levels as determinants of age-dependent lethality.

TABLE 2
Primers used in qRT-PCR analysis purified RNAs from brains of age-matched mock and POWV-infected mice on